Studying the role of anti-capsular antibodies in mediating pathogenesis of carbapenem-resistant Klebsiella pneumoniae

项目摘要/摘要 耐碳青霉烯类肺炎克雷伯菌(CR-KP)引起严重的医院获得性感染 死亡率超过50%，需要新的治疗策略。单抗(MAb)17H12 由Fries实验室开发的结合到胶囊多糖(CPS)上的最突出的 美国的CR-KP克隆，ST258。该单抗已被证明与ST258菌株发生广泛反应。 多种血清型，体外通过吞噬等机制杀伤CR-KP，保护小鼠免受CR-Kp感染。 肺炎克雷伯菌在气管内和肠道外传播模型中的感染。然而，它仍然是未知的 如果17H12要用于治疗，这种单抗如何在体内产生保护，以及这些问题是至关重要的 CR-KP主要影响免疫功能低下的患者。 此外，我们的合作还发现了一种人工合成的寡糖表位17H12 也可以识别并诱导对该ST258亚群的CPS的免疫原性反应。此数据 表明17H12的目标可以作为全尺寸的更便宜、更容易生产的替代方案 CPS还可以涵盖更广泛的菌株，但这种可能性仍有待测试。 本提案旨在确定17H12在体内减轻CR-KP感染的方法，并进行试验 17H12的合成表位可作为潜在的疫苗。我将验证我的核心假设，即抗囊膜 抗体通过专业吞噬细胞的作用保护小鼠免受CR-KP肠道外传播， 而这些单抗识别的表位可以用来防止传播。在《目标1》中，我将 检查抗体介导的保护是否需要吞噬细胞群和Fc受体 相对于补体介导的机制。这一目标将利用各种调查工具，从Flow 流式细胞术和转化子检测哪些常驻吞噬细胞群结合GFP标记的CR-KP 免疫球蛋白亚类开关变异体和Fc受体体外敲除抗体的存在 吞噬试验和体内感染研究。我还会用活体显微镜观察单抗- 实时介导吞噬细胞功能。在目标2中，我将评估接种小分子疫苗是否 17H12识别可以预防ST258分支2 CR-KP的感染。我会确定我们的合成材料 结合17H12的寡糖通过疫苗接种和免疫诱导对全尺寸CPS产生优越的免疫应答 挑战。此外，我将利用噬菌体展示来鉴定模拟CPS表位的多肽，并测试 17H12识别该模拟表位的方式与识别寡糖的方式相同。这项研究的发现 该提案不仅将使我们能够审查抗被膜免疫在清除CR-KP方面的作用，而且还将使我们能够 使我们能够确定哪些患者可能从单抗治疗中受益最大，促进疫苗接种努力，以及更好地 了解ST258克隆的胶囊。

PROJECT SUMMARY/ABSTRACT Carbapenem-resistant Klebsiella pneumoniae (CR-Kp) causes serious hospital-acquired infections with mortality rates exceeding 50%, and new strategies are required to treat it. The monoclonal antibody (mAb) 17H12 developed by the Fries Laboratory binds to the capsular polysaccharide (CPS) to a subset of the most prominent CR-Kp clone in the United States, ST258. The mAb has been shown to broadly react with ST258 strains of multiple serotypes, kill CR-Kp through phagocytosis and other mechanisms in vitro, and protect mice from CR- Kp infection in both an intratracheal and extra-intestinal dissemination model. However, it remains unknown how protection by this mAb occurs in vivo, and such questions are crucial if 17H12 is to be used to treat immunocompromised patients that CR-Kp primarily affects. Furthermore, our collaborations have also uncovered a synthetic oligosaccharide epitope that 17H12 recognizes and can elicit an immunogenic response to the CPS of this ST258 subset as well. This data suggests that the target of 17H12 may serve as a cheaper, more easily producible alternative to full-sized CPS that can also cover a wider range of strains, but this possibility remains to be tested. This proposal seeks to determine the means by which 17H12 mitigates CR-Kp infection in vivo, and test synthetic epitopes of 17H12 that can act as potential vaccines. I will test my central hypothesis that anti-capsular antibodies protect mice from CR-Kp extra-intestinal dissemination through the action of professional phagocytes, and that epitopes that these mAbs recognize can be used to protect against dissemination. In Aim 1, I will examine whether phagocyte populations and the Fc receptor are required for antibody-mediated protection relative to complement-mediated mechanisms. This Aim will utilize a variety of investigative tools, from flow cytometry and transformants to examine which resident phagocyte populations take up GFP-labeled CR-Kp in the presence of antibody, to the use of IgG subclass switch variants and Fc receptor knockouts in ex-vivo phagocytosis assays and in vivo infection studies. I will also apply intra-vital microscopy to observe mAb- mediated opsonophagocytosis in real time. In Aim 2 I will assess whether vaccination with small molecules that 17H12 recognizes can protect against infection by ST258 clade 2 CR-Kp. I will determine whether our synthetic oligosaccharide that binds 17H12 elicits a superior immune response to full-sized CPS through vaccination and challenge. Additionally, I will utilize phage display to identify a peptide mimic of the CPS epitope and test whether 17H12 recognizes this mimotope in the same way as it recognizes the oligosaccharide. The findings of this proposal will not only allow us to examine the role of anti-capsular immunity in the clearance of CR-Kp, but also allow us to identify patients who may benefit best from mAb therapy, promote vaccination efforts, and better understand the capsule of the ST258 clone.